Design, Setting, and Participants Prospective family cohort study to investigate patients at a children's
hospital in Taiwan and family members of these patients who had signs and
symptoms suggestive of enterovirus 71 between February 2001 and August 2002.
Patients and household members underwent clinical evaluations, virological
studies, questionnaire-based interviews, and were followed up for 6 months.

Main Outcome Measures Enterovirus 71 infection, defined as a positive viral culture from a
throat or rectal swab, or the presence of IgM or a 4-fold increase in neutralizing
antibody in serum; and clinical syndromes, defined as asymptomatic; uncomplicated
symptomatic; and complicated; with unfavorable outcomes of sequelae or death.

Results Ninety-four families (433 family members) had at least 1 family member
with evidence of enterovirus 71 infection. The overall enterovirus 71 transmission
rate to household contacts was 52% (176/339 household contacts). Transmission
rates were 84% for siblings (70/83); 83%, cousins (19/23); 41%, parents (72/175);
28%, grandparents (10/36); and 26%, uncles and aunts (5/19). Of 183 infected
children, 11 (6%) were asymptomatic and 133 (73%) had uncomplicated illnesses
(hand, foot, and mouth disease, herpangina, nonspecific febrile illness, upper
respiratory tract infection, enteritis, or viral exanthema). Twenty-one percent
(39/183) experienced complicated syndromes including the central nervous system
or cardiopulmonary failure. During the 6-month follow-up, 10 died and 13 had
long-term sequelae consisting of dysfunction in swallowing, cranial nerve
palsies, central hypoventilation, or limb weakness and atrophy. Age younger
than 3 years was the most significant factor associated with an unfavorable
outcome in children (P = .004). Among 87 infected
adults, 46 (53%) were asymptomatic, 34 (39%) had nonspecific illnesses of
fever, sore throat, or gastrointestinal discomfort, and 7 (8%) had hand, foot,
and mouth disease. There were no complicated cases in adults.

Conclusions Enterovirus 71 household transmission rates were high for children in
Taiwan and severe disease with serious complications, sequelae, and death
occurred frequently. In contrast, adults had a much lower rate of acquisition
of the infection and much less adverse sequelae.

Outbreaks of enterovirus 71 have been documented since it was recognized
in California in 1969.1 Fatalities in adults
and children have been reported in Bulgaria, Hungary, and Malaysia.2- 4 The largest and most
severe enterovirus 71 epidemic occurred in Taiwan in 1998.5- 10 The
clinical syndromes and severity of cases were diverse. Thousands of cases
of hand, foot, and mouth disease (HFMD) and herpangina occurred, 405 experienced
severe neurological complications, such as encephalitis, meningitis, poliolike
syndrome, and encephalomyelitis, and/or pulmonary edema.9 Among
the 405 complications, 78 children died.9

In a seroepidemiological study before and after the 1998 outbreak, we
found that preepidemic and postepidemic enterovirus 71 seroprevalence rates
in adults and children older than 6 years ranged from 57% to 67%.11 The postepidemic enterovirus 71 seropositive rates
among children younger than 3 years old ranged from 0% to 36%11 and
from 26% to 51% for 3- to 6-year-old children. Interestingly, only 29% of
preschool children infected with enterovirus 71 developed HFMD and herpangina.
Another Taiwanese seroepidemiological study12 examined
serial serum antibody titers to enterovirus 71 in blood samples from 81 children
born in 1988 (samples obtained yearly from 1989-1994 and from 1997-1999).
Enterovirus 71 seroconversion occurred with a yearly incidence of 3% to 11%
between 1989 and 1997. By 1997, 68% of the 81 children had serological evidence
of enterovirus 71 infection.12 A seroepidemiological
study in Singapore also demonstrated that the enterovirus 71 seroprevalence
rate in the general population was as high as 60% to 70%.13 It
is evident that enterovirus 71 infection is not uncommon and that documented
cases do not accurately reflect the actual number of infections.

The reason why the 1998 enterovirus 71 outbreak in Taiwan was so large
is not clear.9,10 Enterovirus
71 seropositivity was found to be concordant among siblings in our previous
seroepidemiological study, suggesting that household transmission may play
an important role in the spread of enterovirus 71.11 The
secondary household transmission rates of enteroviruses, such as poliovirus,
enterovirus 70, and coxsackievirus A24, vary.14- 16 Transmission
of enterovirus 71 among household family members needs further investigation.
Such data are necessary to help control, manage, and prevent future enterovirus
71 infections. Therefore, the objectives of this study were to investigate
enterovirus 71 transmission and determine clinical outcomes within households
of patients infected with enterovirus 71.

METHODS

At Chang Gung Children's Hospital in Taiwan, we studied patients who
were suspected of having enterovirus 71 illnesses, such as HFMD and herpangina,
and their household family members between February 2001 and August 2002.
Institutional review board approval was obtained from the Chang Gung Memorial
Hospital for this study and informed consent was obtained from all patients
or their parents.

Patients presenting to the emergency department, outpatient clinic,
or inpatient ward who had clinical syndromes suggestive of enterovirus 71
infection were asked to participate in the study along with their household
family members. Throat and rectal swabs or stool samples for virus isolation
and a blood sample for enterovirus 71 IgM neutralizing antibodies were obtained
from the suspected cases of enterovirus 71 treated at Chang Gung Children's
Hospital. Clinical manifestations, disease course, and outcomes were recorded.
Family members in the same household were asked to undergo screening with
a throat swab and blood sample.

Questionnaire-based interviews were used to collect information about
the family members, including demographic data, the number of bedrooms in
the house, amount of contact time with the patient, presence and pattern of
current or recent signs and symptoms (ulcers, sore throat, rash, fever, abdominal
pain, and diarrhea), and contact history with persons outside of the household
who had clinical syndromes suggestive of enterovirus 71 infection. Follow-up
telephone interviews repeated questions about signs and symptoms at 2, 4,
and 8 weeks. If any household family member reported experiencing signs or
symptoms suggesting enterovirus 71 infection during the follow-up period,
clinical assessment and laboratory investigation for enterovirus 71 were repeated.
Patients with complications were followed up for 6 months at the hospital
as outpatients or inpatients.

If the suspected case or any household family member tested positive
for enterovirus 71, a second blood sample was obtained from the suspected
case and household family members 4 weeks after the first blood sample. Household
enterovirus 71 transmission rates and clinical outcomes were analyzed only
for families with at least 1 member with evidence of enterovirus 71 infection.

Clinical Syndromes, Outcomes, and Identified Source of Infection

Laboratory evidence of enterovirus 71 infection was defined as the isolation
of enterovirus 71 from a throat or rectal swab or stool sample, or the presence
of enterovirus 71 IgM, or a 4-fold increase in enterovirus 71 neutralizing
antibody serotiters between acute and convalescent sera samples.

In uncomplicated cases, evidence of HFMD included oral ulcers on the
tongue and buccal mucosa and a vesicular rash on the hands, feet, knees, or
buttocks. Evidence of herpangina included oral ulcerations on anterior tonsillar
pillars, soft palate, buccal mucosa, or uvula. Nonspecific febrile illness
was defined as a rectal temperature greater than 38°C without other symptoms.
Enteritis was defined as diarrhea with or without abdominal pain. Upper respiratory
tract infection was defined as sore throat, coryza, or cough without herpangina
or rash.

In complicated cases, aseptic meningitis was defined as a clinically
compatible illness with cerebrospinal fluid pleocytosis (>5 leukocytes/mm3 in patients >1 month or >25 leukocytes/mm3 in neonates)
and negative bacterial cultures. Encephalitis was characterized by an altered
level of consciousness accompanied by cerebrospinal fluid pleocytosis. Evidence
of a poliomyelitislike syndrome included acute limb weakness with diminished
reflexes and muscular strength. A diagnosis of encephalomyelitis was made
when there was evidence of encephalitis and poliomyelitislike syndrome. Cardiopulmonary
failure was defined as pulmonary edema and hemorrhage with left ventricular
failure requiring inotropic support. Unfavorable outcome was defined as death
or sequelae and favorable outcome was defined as complete recovery after 6
months of follow-up.

The index cases were the first members of the household to have clinically
apparent illness confirmed by laboratory studies. The secondary cases were
defined as other family members whose enterovirus 71 symptoms occurred later
than the index cases' illness. Identified source of enterovirus 71 infection
within the household was defined as the first case in the household who displayed
clinically apparent disease and who had had clear contact history with individuals
outside the household who had illnesses suggestive of enterovirus 71 infection,
such as HFMD and herpangina. The infection transmission interval was defined
as the time between the onset of disease for the first case in the household
and the onset of disease in a secondary case. A crowded household was defined
as the ratio of the number of household members to the number of bedrooms
greater than 1.5.

Laboratory methods for measuring enterovirus 71 neutralizing antibody
followed standard protocol for the neutralization test on microtiter plates.17,18 Serum and 50 µL of enterovirus
71, containing 100 fifty percent tissue culture infective doses of enterovirus
71 strain TW/2272/98 (GenBank accession number AF119795), were mixed and incubated
onto the microtiter plates with rhabdomyosarcoma cells at 35°C in a 5%
carbon dioxide incubator. Each tested sample was run simultaneously with cell
control, serum control, and virus back titration. Cytopathic effect was observed
under an inverted microscope after an incubation period of 2 to 7 days, and
the neutralizing antibody titer was determined at the time when cytopathic
effect was observed in 1 fifty percent tissue culture infective dose of
the virus back titration. The neutralizing antibody titer was defined as the
highest dilution of serum that would prevent the occurrence of cytopathic
effect. Seropositivity was defined as a neutralizing antibody titer of 8 or
higher.

Enterovirus 71 IgM Detection

Enterovirus 71 isolate TW/2086/98 was amplified and purified as an antigen
for m-capture enzyme-linked immunosorbent assay. Compared with the standard
method of conventional virus culture, the sensitivity for µ-capture
enzyme-linked immunosorbent assay was 91.5% and the specificity was 93.1%.19

Statistical Analysis

Data were analyzed using SAS statistical software (Version 8.2, SAS
Institute, Cary, NC). We used the t test for continuous
variables and the χ2 test for categorical data. Univariate
analysis was used to screen for statistically significant variables; then
forward stepwise multiple logistic regression analysis was performed to adjust
for confounders simultaneously and to calculate multivariable adjusted odds
ratios (AORs) for risk factors of enterovirus 71 infection and an unfavorable
outcome in children. The relative risk (RR) was calculated using the formula
of Zhang and Yu,20 which corrects AORs obtained
from logistic regression if the incidence of an outcome is higher than 10%
in the study subjects. The α level of model selection was set at .15
for in and out models. P<.05 was considered statistically
significant.

To adjust individual results within families, the PHREG program in SAS
with STRATA option was used for conditional logistic regression analysis.
Because the results were similar, only the stepwise multiple regression analysis
results are reported.

RESULTS

One hundred seventy-three suspected cases of enterovirus 71 and their
household members (343 children and 441 adults) were investigated between
February 2001 and August 2002 (Figure 1).
All eligible cases were included; 3 household members (2 fathers and 1 grandfather)
refused to participate. Ninety-four families (54%) had at least 1 member with
enterovirus 71 isolation and were studied futher. The source of infection
to the 94 families was only identified in 44 families (47%) (19% from relatives
outside of the household, 13% from schoolmates, 11% from neighbors, and 4%
from friends).

The enterovirus 71 infection transmission interval ranged from 1 to
15 days. The median transmission interval was 3 days and the mean (SD) interval
was 3.7 (2.6) days. The enterovirus 71 transmission rate was 52% (176/339)
among household contacts, including an 84% transmission rate for siblings
(70/83); 83% for cousins (19/23); 41% for parents (72/175); 28% for grandparents
(10/36); and 26% for uncles or aunts (5/19) (Table 1). The transmission rate was 84% (89/106) among household
children and 37% (87/233) among household adults (P<.001).
The 39% (41/106) culture-positive enterovirus 71 rate from household children
was also significantly higher than the 4.3% (10/233) culture-positive enterovirus
71 rate from household adult contacts (P<.001).
Among children, no significant difference in the infection rate existed between
siblings and cousins. Among adults, parents had a higher infection rate (41%;
72/175) than other adults (26%; 15/58) (P = .05).
Infection rate for mothers (43%; 40/92) was similar to that for fathers (39%;
32/83) (P = .61). Enterovirus 71 seropositive rates
for all family members were as high as 93% (401/429).

Enterovirus 71 infection rates declined as age increased (Table 2). Furthermore, all 71 children who were younger than 2 years
were infected with enterovirus 71.

Factors Associated With Enterovirus 71 Infection in Children

Male sex and age younger than 6 years were associated with an increased
risk of enterovirus 71 infection (Table
3). Children attending kindergarten or school had a lower incidence
of enterovirus 71 infection. Having more household members, more children
in the household, and a more crowded household did not significantly increase
the risk of infection. Forward stepwise multiple logistic regression analysis
indicated that the most significant factors associated with infection in children
were age younger than 6 years (AOR, 9.11; 95% confidence interval [CI], 2.90-28.65; P<.001 and the corrected RR [CRR], 2.37; 95% CI, 1.74-2.68)
and male sex (AOR, 4.11; 95% CI, 1.19-14.15; P =
.03 and CRR, 1.13; 95% CI, 1.02-1.19).

Clinical Syndromes and Outcomes in Children

Children had significantly higher rates than adults of complications
(21% vs 0%, P<.001) and long-term sequelae and
fatalities (13% vs 0%, P = .001) (Table 4). The clinical outcome of the secondary cases was not significantly
different from that of primary cases (P = .09).

Of 183 children, 10 (5%) died: 6 patients within 24 hours of hospitalization
due to brainstem encephalitis plus fulminant cardiopulmonary failure; 3 patients
within 2 to 7 weeks of hospitalization due to brainstem encephalitis plus
deep coma; and 1 patient with sequelae of dysphagia plus central hypoventilation
at 4 months due to home ventilator dysfunction.

Based on univariate analyses, age younger than 3 years was the most
significant factor associated with long-term sequalae or death (Table 5). Contact history with a person outside the household with
HFMD and hepangina, more household members, more children in the household,
and living in a crowded household were not associated with a significantly
higher unfavorable outcome rate. Children in kindergarten and school had lower
unfavorable outcome rates. Forward stepwise multiple logistic regression analysis
indicated that the most significant factor associated with an unfavorable
outcome in infected children was age younger than 3 years (AOR, 6.19; 95%
CI, 1.77-21.6; P = .004 and CRR, 5.18; 95% CI, 1.72-12.2),
but there were no deaths in infants younger than 3 months.

In this prospective family cohort study, we found that enterovirus 71
infections in young children are associated with serious disease; we also
found a high household transmission rate for children. In contrast, infection
in adults was less frequent and severe. Long periods of viral shedding may
account for widespread transmission of enterovirus 71.14 In
a previous study, we found enterovirus 71 to be present in infected patients
for up to 5 weeks (based on stool sample analysis).21 Alternatively,
previous research has demonstrated a higher rate of enterovirus 71 isolation
from throat swabs (90%) than from rectal swabs or stool samples (32%).22 We speculate that respiratory transmission by large
droplets from the oral cavity may also explain the high secondary infection
rate within households in Taiwan, despite hand-washing precautions in practice
since 1998.23 Therefore, isolation of infected
patients within single rooms with masks for the patients and close contacts
should be considered for the prevention of transmission of enterovirus 71.

New York Virus Watch data indicate that secondary coxsackievirus infections
are more frequent in mothers (78%) than fathers (47%).15 However,
in this study, enterovirus 71 infection rates were similar in the mothers
(43%) and fathers (39%). Enterovirus 71 infection rates for parents (41%;
72/175) were higher than for other adults (26%; 15/58), suggesting that close
or longer contact facilitated enterovirus 71 transmission.

Enterovirus 71 seropositive rates for all family members were as high
as 93% (Table 1), which were significantly
higher than those (57%-67%) for the general population in a previous seroepidemiological
study.11 Therefore, it is likely that almost
all the susceptible family members were infected once enterovirus 71 had been
introduced in the household. Because the enterovirus 71 infection rate increased
as age decreased, the most susceptible households with the youngest members
had the highest enterovirus 71 infection rates. The high level of infection
with enterovirus 71 is similar to that of poliovirus.14

Among children, household transmission produced a higher rate of clinical
symptoms (94%) compared with transmission outside the household (29%) in our
previous enterovirus 71 seroepidemiological study.11 Viral
load or host genetic factors may account for this difference. Because the
rate of asymptomatic infection with enterovirus 71 after social contact is
high (about 71%),11 it was difficult to identify
the source of primary infections to the family. We were successful in determining
the source in only 47% of cases.

Enterovirus 71 infections in adults were less serious than those in
children. Although enterovirus 71 infection should be considered in cases
of adult encephalitis, unexplained pulmonary edema, or cardiopulmonary failure,
most infected adults in the present study were asymptomatic or experienced
mild upper respiratory tract infections.2,24 The
reasons why adults or older children have less severe illness needs further
study. One possible reason for reduced severity might be that most adults
and older children have antibody to enterovirus 71 or a related virus, whereas
younger children may incur primary infections. Another possible reason is
that inadequate hygiene of younger children may increase the viral load and
produce more severe disease. Enterovirus 71 transmission by infected adults
who are asymptomatic or mildly symptomatic is a likely source of many infections.
In some families whose source of infection could not be identified, we speculate
that enterovirus 71 infections might be introduced into the family by an asymptomatic
adult. The transmission rates and outcomes of cases infected by contact with
an asymptomatic case is unknown.

In conclusion, enterovirus 71 household transmission rates were high
for children in Taiwan. In addition, enterovirus 71 was associated with serious
complications, long-term sequelae, and death in children younger than 3 years.
Adult enterovirus 71 infection was usually asymptomatic or mild.